1. Field of the Invention
The present invention relates to an optical bio-imaging technique using fluorescence detection.
2. Description of the Related Art
The method for attaining imaging as to what state a molecular species in a living body or in cells is distributed into is an important researching method in medical science or biology. At the level of cells, the following method has widely been performed: a method of using a microscope to image a molecular species with a molecular probe to which a fluorescent dye adheres or a gene-expression molecular probe. Regarding those which are larger than cells, such as internal organs, and further, animal individuals, a device has been required for observing considerable molecular species and the state of their distribution while alive.
The method is, for example, a technique of bonding a fluorescent probe to a cancer cell in an individual of a mouse or some other, imaging the situation of the proliferation of the cancer cell, to which attention is paid, and observing the situation every day or every week to analyze a change in the situation with the passage of time. In order to watch the proliferation of cancer cells inside an individual of an animal by means of a conventional cell-level-observing measuring device, the animal is killed and then a predetermined region thereof is dyed or a fluorescent substance is attached thereto so as to observe the region. However, according to the method, it is impossible to watch the proliferation of cells of a single individual with the passage of time over a long term. For this reason, it has been desired to develop a device capable of observing a molecular species of an individual of a small animal, as internal information thereof, in the state that the individual is alive.
FIG. 10 is a view illustrating an example of a typical fluorescence imaging apparatus.
According to this device, out of light rays from a light source 16, light rays having wavelengths selected through an excitation-side filter 11 (Fex) are radiated, as excitation light, onto a living sample, and then fluorescent components of scattered light rays therefrom are taken out through an emission-side filter 12 (Fem) so as to form an image on a CCD camera 38, which is a two-dimensional detecting unit, through an imaging lens 32. In this way, a fluorescent image of the sample is obtained.
In such a device, at the time of radiating excitation light onto a sample, its fluorescent molecule to which attention is paid emits light having a wavelength different from that of the excitation light, normally, light having a wavelength longer than that of the excitation light; thus, when a filter which blocks the wavelength components of the excitation light completely is set up as the emission-side filter 12 between the sample and the two-dimensional detecting unit 38, only the fluorescent wavelength components can be detected with a good sensitivity.
Actually, however, the spectrum of the excitation light radiated onto the sample slightly contains therein light having the same wavelengths as the fluorescent components (the light may also be referred to as stray light) in many cases. The stray light is reflected on the sample, and overlapped with the fluorescence emitted from the sample so as to deteriorate the detection limit of the fluorescence. If the excitation light radiated onto the sample does not contain stray light at all, the following drawback is caused when the emission-side filter is insufficient in capability so as not to remove the wavelength components of the excitation light completely: components of the excitation light reflected on the sample are partially transmitted through the emission-side filter so as to overlap with the fluorescent components from the sample, so that the detection limit of the fluorescence is deteriorated. The deterioration in the fluorescence detection limit causes feeble fluorescent components to be buried in noises, so that the fluorescent components cannot be vividly imaged. In a case where at the time of causing a fluorescent dye to adhere onto a notable region of a living sample and observing the region the notable region is present around a central position of the living sample, that is, around a position farther from the surface thereof, the intensity of the fluorescence captured from the surface of the living sample becomes weak accordingly. When the fluorescence detection limit is bad, such feeble fluorescent components cannot be vividly captured.